Aflatoxins (AFs) are among the most carcinogenic and teratogenic secondary metabolites (SMs) produced by the human pathogen Aspergillus flavus and A. parasiticus. These ubiquitous fungi infect our food products and crops and produce AFs which, upon ingestion, form AF/DNA adducts, impair immunity and cause liver cancer. Since it is impossible to eliminate the AF-producing fungi from nature, there is a great need to address the problem of AF contamination. The major goal of this study is to expand our knowledge n the field of genetic regulation of AF production. Recently, LaeA, a nuclear protein and a global regulator of secondary metabolism, was discovered in A. nidulans, a related fungus making sterigmatocystin (ST, a late precursor of AF) as its SM. In ongoing studies, we have isolated the laeA ortholog in the AF-producing A. flavus. Deletion of laeA results in loss of AFs, reduced infectivity and loss of sclerotia (developmental structures associated with AF production). These characteristics overlap with our experimentally induced AF-negative, developmentally altered A. parasiticus sec- (for secondary metabolism minus) variants. In this proposal we will test three hypotheses: (1) The AF-negative sec- phenotype involves changes in laeA expression. To test this hypothesis, we will carry out experiments to both over-express and delete laeA in the sec- strains. (2) LaeA exerts its effects on AF production by interacting with AF pathway-specific regulators and/or with other chromatin proteins. To test this hypothesis, we will use yeast two-hybrid assays. (3) The sec- phenotype is caused by epigenetic events. The sec- strains, which have bypassed certain developmental events, are more likely at risk for chromosomal damage, especially in the sub-telomeric region, a region which harbors the AF cluster and is susceptible to genetic abnormalities. To test for epigenetic changes, we will examine changes in DNA methylation and transposon position in sec- mutants. Results from these studies will better define the role of global transcriptional regulators (LaeA) on AF production and secondary metabolism in general. Long-term, this may assist researchers to develop better strategies (e.g., identification of natural inhibitors and development of genetically engineered strains) to reduce AF contamination. Knowledge from this study may also have a broader impact on two areas characterized by abnormal development, chromosomal aberrations and epigenetic changes;namely, aging and cancer.